Transplanted cells provide the potential for treating various diseases because of their ability to detect and respond to physiologically important substances in the host. Cell implantation therapy is particularly desirable because the cells can provide substances to replace or supplement natural substances which, due to their insufficiency or absence cause disease. The release of therapeutic substances from the transplanted cells may also be properly regulated provided the transplanted cells have the necessary receptors and ability to respond to endogenous regulators.
Patients having disease as a result of the loss or deficiency of hormones, neurotransmitters, growth factors or other physiological substances are considered to be among those who would achieve significant benefits from transplant therapy. For example, implantation of pancreatic islet cells could provide insulin as needed to a diabetic. Adrenal chromaffin cells or PC12 cells implanted in the brain may provide dopamine to treat patients with Parkinson's disease. Several other hormones, growth factors and other substances have been identified and are discussed in PCT application WO 92/19195 (which is incorporated herein by reference), as potential therapeutics which could be administered to an individual using transplanted cells.
Because cells which are implanted may be foreign to the host it is necessary to prevent the host immune system from attacking and thereby causing the death of the implanted cells. In addition, cells which secrete such therapeutic substances may have been derived from transformed cells or have been infected with viruses and may therefore present a potential threat to the host in the form of increasing the likelihood of tumor formation. At least four methods are possible to attenuate the host immune response for the purposes of protecting the transplant cell viability. One method involves immunosuppression to prevent transplant rejection. Immunosuppression may be accomplished through a variety of methods, including using immunosuppressive drugs such as cyclosporins. In another method, immunomodulation, the antigenicity of the implanted cells is altered. This could involve attaching antibody fragments to the implanted cells. The third method involves modulating the host immune system to obtain tolerance to the implanted cells. In a fourth method, the cells to be implanted are contained in a device which effectively isolates the implanted cells from the immune system. The ability of contained cells to manufacture and secrete substances of therapeutic value has led to the development of implantable devices for maintaining cells within an individual in need of treatment.
A common feature of isolation devices is a colony of living cells surrounded by a permeable membrane. The transport of nutrients, waste and other products across the membrane is driven by pressure and/or diffusion gradients. This movement of substances across the membrane is limited by the permeability of the membrane and the distance through which these substances must travel. If insufficient transport of these substances is provided for either the number or volume of cells, cell viability and function may be diminished.
Dionne, has reported that a dense metabolically active cell mass must not exceed certain maximum dimensions if the viability of the entire cell mass is to be maintained. "Effect of Hypoxia on Insulin Secretion by Isolated Rat and Canine Islets of Langerhans", Diabetes, Vol. 42, 12: 20, (January 1993). When large spheroidal cell agglomerates receive nutrition from an external source, cells at the center of the cell mass may not receive sufficient nutrition and die.
Most encapsulation devices feature larger cell chambers that which will allow diffusion of a sufficient flux of nutrients to support a viable full density cell mass. A full density cell mass is the maximum number of cells which can be maintained in a fixed volume if the entire space available for cells is occupied by the cells to achieve a minimum of cell-free space. This number is approximated by dividing the total available volume for containing cells by the volume of a single cell.
In the cylindrical devices referred to by Aebischer in Wo 92/19195, the diameter is larger than the maximum diameter which would support a viable full density cell mass. Accordingly, the cells of the device described in WO 92/19195 must be in a diluted suspension at a lesser cell density. The diluted cell suspension has lower overall nutrient requirements per unit volume and thus maintains essentially full viability with the available nutrient transported through the permeable membrane. The larger than optimum cell container allows for easier manufacture and subsequent manipulation than would be possible if this device were made small enough to support an optimum, full density cell pack. Aebischer also refers to the use of a gelling substance in the cell suspension to immobilize the cells into a uniform dispersion to prevent aggregation of cells into clumps. Such clumps could otherwise become necrotic due to localized depletion of nutrients within these clumps.
Several immunoisolating devices have been developed for implanting cells in a host. U.S. Patent 5,158,881, refers to a device in which cells are stated to be encapsulated within a semipermeable, polymeric membrane by co-extruding an aqueous cell suspension of polymeric solution through a common port to form a tubular extrudate having a polymeric outer coating which encapsulates the cell suspension. In one embodiment described in the U.S. Pat No. 5,158,881, the cell suspension and polymeric solution are extruded through a common extrusion port having at least two concentric bores, such that the cell suspension is extruded through the inner bore and the polymeric solution is extruded through the outer bore. The polymeric solution is stated to coagulate to form an outer coating. In another embodiment of the U.S. Pat. No. 5,158,881 patent, the tubular extrudate is sealed at intervals to define separate cell compartments connected by polymeric links.
A different approach to supply nutrients to an isolation device is to route a flowing blood supply or other physiologic fluid through one or more conduits within the cell mass. This internalized source of nutrient mimics the structure of the circulatory system of almost all complex organisms, by providing nutrient to the center of a cell mass or tissue. These nutrients then diffuse radially outward. In one such internally fed device described in WO 91/02498, the transplanted cells are contained in-between two concentric tubes. One end of the inner tube is grafted to an artery while the other end is grafted to a vein. A common problem with internally fed devices is the potential for thrombosis formation or clotting of blood within the artificial conduits which occurs in relatively short periods of time. The formation of such obstructing masses cut off the flow of nutrients to internally fed devices.
In another device described by Goosen, U.S. Pat. Nos. 4,673,566, 4,689,293 and 4,806,355, the cells are contained in a semisolid matrix which is encapsulated in a biocompatible semipermeable electrically charged membrane. The membrane is stated to permit the passage of nutrients and factors while excluding viruses, antibodies and other detrimental agents present in the external environment.
WO84/01287 refers to devices for encapsulating genetically programmed living organisms. One of the devices referred to comprises a nutrient material surrounded by an inner membrane wall which is surrounded by a layer of organisms surrounded by an outer membrane wall. The organisms are stated to provide therapeutic substances. These organisms receive nutrients from the inner layer. Including a nutrient layer in the center of the device makes the manufacture of such devices difficult and expensive.
For implanted devices to be therapeutic, enough cells must be present and viable within the device to manufacture and secrete therapeutically effective amounts of a therapeutic substance. If too many cells are consuming nutrients within the device, the local concentration of these solutes will drop below the minimum level required for cell viability. Cells which are located near the outer surface of the cell mass will typically receive ample nutrition, while cells located in the interior will be the first to die or otherwise become disabled. Factors which may have a negative effect on the viability of cells contained within a device are: device dimensions which position cells far from nutrients; cells with high metabolic demand; and any resistance to diffusive transport resulting from thick or impermeable membranes or unstirred fluid layers. Cell masses which become too large may inhibit diffusion of nutrients or gasses into the depths of the cell mass, resulting in the death of such cells and a correspondingly decreased substance output. This phenomenon is reported by Schrezenmeir, et al., in "The Role of Oxygen Supply in Islet Transplantation", Transplantation Proceedings, Vol. 24, No. 6, pp. 2925: 2929, (1992), which reports a central core of necrosis in islets greater than about 150 microns diameter after culturing of the islets in an encapsulating device. Additionally, the secretion of other factors associated with lysis of dead cells may be harmful to the host or adjacent cells.
Another method for maintaining the viability of cells within an encapsulating device is to make the device sufficiently narrow to keep the cells sufficiently close to the permeable membrane in contact with the environment. Decreasing the device diameter however, results in a finer, more fragile structure which is increasingly hard to manufacture and use.
Another requirement of encapsulation devices is that the device must have sufficient mechanical strength and a geometry suitable for allowing the device to be manipulated by a surgeon during implantation. Mechanical integrity allows immunoisolating devices to be manipulated as a unit. Strength requirements will vary with the size, weight and shape of the device, but in general, the longer, heavier, or larger the device, the stronger it will have to be. As the number of cells required to provide therapeutic benefit increases, the size of the device and the amount of structural material must also increase. The additional structural material necessary to manufacture a larger device may interfere with the function of the device if it reduces cell viability or the transport of therapeutic substances. Suitable geometries for implantation might include a size and shape which can be handled aseptically using gloved hands and surgical instruments, and which will fit in the intended implant sites within the host.
Various methods have been described for filling devices with living cells. Some devices are filled after the permeable membrane is formed by flushing a cell suspension into the device, while other devices are produced by forming a membrane around the cell mass using a chemical process which causes the membrane to form without killing cells. In the former, the device is easier to load with viable cells if the dimensions of the device are large enough to allow low shear flow of the cell suspension. In the latter example, larger device dimensions also enhance manufacturability as a greater proportion of cells remain viable because they are protected from the membrane formation process by the presence of an unstirred fluid layer and are more distant from the site of membrane formation. A disadvantage of larger device dimensions occurs when cells near the surface are triggered to respond to a stimulus which may not reach cells situated more internally therefore diminishing the release of the therapeutic substance from the internally situated cells.
It is therefore necessary to develop a device of suitable geometry and strength which can provide an adequate number of viable cells and which may be inserted in an individual.